HIV infection is characterized by a progressive decline in immune system function, suppressing the infected host's ability to overcome other, secondary infections. No cure has been found for HIV infection. The pathogenetic process in HIV infection is never unidimensional but, rather, extremely complex and multifactorial. The pathogenic progression may be only tangentially related to the direct infection of a given target cell. Fauci, 1993, Science 262: 1011-1018. Death is inevitable, usually from an overwhelming secondary infection and/or HIV related neoplasm.
Current treatments for HIV infection attempt to retard the progress of the disease or relieve symptoms. Treatments in use today include certain dideoxynucleosides such as azidothymidine (AZT or zidovudine, Burroughs-Wellcome), dideoxyinosine (ddI, Bristol-Myers Squibb) or dideoxycytidine (ddC, Hoffman-LaRoche). These agents can be toxic. Their applicability is limited because of the appearance in some patients of onerous, and sometimes lethal, side effects. These side effects include myelosuppression, peripheral neuropathy, and pancreatitis. In some patients, AZT has lost its effectiveness after prolonged use. While many other drugs have been proposed for treatment of HIV infection, none have been demonstrated to be effective.
Defibrotide is a polyanion salt of a deoxyribonucleic acid obtained from mammalian tissue. Defibrotide is a single-stranded polydeoxyribonucleotide with molecular weight of approximately 20 kDa (low molecular weight form) which may be obtained from bovine lung DNA by controlled hydrolysis. Patents related to its manufacture include U.S. Pat. No. 3,770,720 directed to a process for extracting DNA from mammalian tissue, and U.S. Pat. No. 3,899,481 directed to a process for the controlled partial degradation of DNA extracted from animal organs.
Defibrotide is noted primarily for its profibrinolytic effects (Pescador et al., 1985, Thromb. Res., 30: 1-11). Defibrotide increases the release of tissue-type plasminogen activator (t-PA) and decreases plasminogen activator inhibitor (PAI1) activity. The increase in t-PA is in conjunction with the decrease in PAI1, the latter being the more prominent action. The profibrinolytic activity of defibrotide is likely due to a decrease in PAI1 levels rather than to an increase in t-PA level (Pogliani et at., 1987, Farmaci E Therapia IV, (2): 1-5; Ulutin et al., International Scientific Symposium on Fibrinogen, Thrombosis, Coagulation and Fibrinolysis, Aug. 30-Sep. 1, 1989, Taipei, Taiwan, ROC).
U.S. Pat. No. 3,829,567 is directed to the use of an alkali metal salt of a polynucleotide or an oligonucleotide of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) as a fibrinolytic agent. U.S. Pat. No. 4,649,134 is directed to a method of treating acute renal insufficiencies accompanied by thrombotic microangiopathy with defibrotide. Such pathologies include hemolytic uremic syndrome (HUS), collagenopathies (e.g., panarteritis and lupus), Wegner, Schoenlein-Henoch, disseminated intravascular coagulation (DIC), fast evolving glomerulonephritis, and thrombotic thrombocytopenia purpura (TPP). U.S. Pat. No. 4,693,995 is directed to a method of treating acute states of myocardial ischemia and infarction with defibrotide.
While the primary target cell of defibrotide action has been shown in numerous studies to be the vascular endothelial cell (Bilsel et al., 1990, Thromb. Res., 58: 455-460), cytotropic actions have been shown for hepatic and myocardial cells as well (Lobel and Schror, 1985, Naunyn-Schmiedeberg's Arch. Pharmacol., 331: 125-130).
Defibrotide has been found to be a prostaglandin I.sub.2 (PgI.sub.2) secretory agent (Niada et al., 1982, Pharmacological Res. Comm., 14: 949-957). Defibrotide also induces synthesis of other prostanoid metabolites, such as prostaglandin E.sub.2 (PgE.sub.2). The increase in secretion of the prostanoid metabolites, in particular PgI.sub.2 and PgE.sub.2, from vascular endothelial cells seems to involve interaction with arachidonic acid metabolites (Costantini et al., 1989, Eur. J. Int. Med., 1: 115-120). It has been shown in rabbits that prostanoid neosynthesis induced directly by arachidonic acid was significantly enhanced by the stimulation of adenosine A.sub.1 and A.sub.2 receptors by defibrotide, especially at those levels that do not directly affect the output rate of PgI.sub.2 and PgE.sub.2 from the rabbit aorta vascular endothelial cells (Ulutin et al., International Scientific Symposium on Fibrinogen, Thrombosis, Coagulation and Fibrinolysis, Aug. 30-Sep. 1, 1989, Taipei, Taiwan, ROC). PgI.sub.2 and PgE.sub.2 promote microcirculatory vasodilation and antagonism of platelet aggregation. Defibrotide induces an in vivo increase in platelet cyclic adenosine monophosphate (cAMP) levels resulting in aleaggregation of platelets and plasma prostanoid levels, as shown in humans (Cizmeci, 1986, Haemostasis, 16 (suppl. 1): 31-35). Defibrotide does not increase levels of malonylaldehyde, thromboxane A.sub.2, thromboxane B.sub.2, .alpha..sub.2 -antiplasmin, or .alpha..sub.2 -macroglobulin activities.
Defibrotide is also known to exhibit antithrombotic actions (Niada et al., 1981, Thromb. Res., 23: 233-246). Defibrotide has been shown to elevate Protein CA and Protein CI levels, which affects antithrombotic action. The reported elevations in the levels of Protein CA and Protein CI are proposed to be via defibrotide's modulatory effects on the vascular endothelial cell-thrombomodulin levels. At the dose levels utilized thus far, it is devoid of anti-coagulant effects (Coccheri et al., 1982, Int. J. Clin. Pharm. Reg., 11(3): 227-245), and no clinical applicability as an anti-coagulant agent has been taught heretofore.
Defibrotide exhibits a synergistic action with, and potentiates the effect of heparin (Ulutin et al., International Scientific Symposium on Fibrinogen, Thrombosis, Coagulation and Fibrinolysis, Aug. 30-Sep. 1, 1989, Taipei, Taiwan, ROC). The synergistic mechanism between defibrotide and heparin is not totally clear. One proposed theory is that defibrotide competitively binds with heparin receptors, promoting prolonged circulation of endogenous heparin. Ulutin et al. reported an increase in anti-Factor Xa activity (Ulutin et al., International Scientific Symposium on Fibrinogen, Thrombosis, Coagulation and Fibrinolysis, Aug. 30-Sep. 1, 1989, Taipei, Taiwan, ROC). This effect may add to its antithrombotic action.
In a comparable animal model of the rat aortic strip, defibrotide was shown to inhibit endothelin-induced contraction of the vascular smooth muscle (Fareed et al., 1990, In: Advances in vascular pathology, Elsevier Science Publishers B. V., pp. 171-177). This implies that factors other than impaired fibrinolysis were being treated by defibrotide, such as defibrotide-induced suppression in the levels of the vasoactive amines secreted from the vascular endothelial cells in response to injury. In umbilical vein human endothelial cell cultures, defibrotide was shown to increase cell number and protein content in the culture supernatant, implying a greater role in translation than in the induction of mitotic activity (Bilsel et al., 1990, Thromb. Res., 58: 455-460).
Additional data on defibrotide reveals that defibrotide can modulate lipid peroxidation of membrane phospholipids and oxygen radical induced inhibition of the cyclooxygenase pathway, two major mechanisms in the process of vascular endothelial cell injury. Analogous to these are defibrotide-induced inhibition of superoxide generation by neutrophils induced from platelet activating factor (PAF) (Cirillo et al., 1991, Haemostasis, 21: 98-105). Defibrotide has shown protective effect in mice against pulmonary embolism, analogous to the free radical scavenging enzymes superoxide dismutase and catalase (Niada et al., 1986, Haemostasis, 16 (suppl. 1): 18-25; Bonomini et al., 1985, Nephron, 40: 195-200). Defibrotide-based antithrombotic action in pulmonary embolism may be analogous to the antioxidant effects of cardiovascular drugs.
The cytotropic effects of defibrotide are proposed to be on the basis of PgI.sub.2 -induced vasodilatation of the microvasculature and the secondary increases in the tissue oxygenation and nutrition. While it has been reported that defibrotide acts via modulation of vascular endothelial cells, its recently formally adopted pharmaceutical classification as a "polypharmaceutical agent" is uniformly ascribed to defibrotide's PgI.sub.2 secretory action.
Pre-clinical and clinical experience with defibrotide as well as ex vivo and animal studies done over the past ten years in Europe evidences a cyto-protective effect in myocardial warm and cold ischemia (increased tissue ATP, ADP, 2-3DPG, NADP/NADPH levels), and in reperfusion injuries in the ischemic myocardium and liver (decreased lactate, CPK, intracellular pH), as well as organ procurement and transplantation, proving its cyto-protective effects in other cell types such as myocardial and. hepatic cells (Niada et al., 1986, Haemostasis, 16 (suppl. 1): 18-25; Berti et al., 1990, Advances in Prostaglandin, Thromboxane, and Leukotriene Research, 21: 939-942). The anti-ischemic effect-induced salvage of the cellular energy pools were ascribed to adenosine receptor induced stimulation of adenylate cyclase enzyme pathway.
Defibrotide therapy has been used in disease states in which inappropriate production or intravascular deposition of fibrin has been a prominent factor. Peripheral obliterative vascular disease (POVD) comprises its primary commercial application in Europe (Ulutin, 1988, Semin. Thromb. Hemost., 14(suppl 1): 58-63), accompanied by the secondary clinical indications of prophylaxis of perisurgical deep vein thrombosis (DVT), and by its less well established use in hemodialysis. Clinical application has been investigated in vasculitides (Raynaud's disease (humans)), prolongation of graft survival in renal transplantation (humans), DIC (animal models), sepsis (animal models), stroke (animal models), renal failure and thrombotic microangiopathy (HUS, TTP (humans)) (Bonomini et al., 1985, Nephron, 40: 195-200; Vangelista et al., 1986, Haemostasis 16 (suppl. 1): 51-54; Oral et al., 1989, Blood 74(suppl 1): 4111a). Defibrotide has been administered to humans primarily as an investigational agent in the United States.
Defibrotide is manufactured by CRINOS Farmacobiologica S.p.A., Villa Guardia (Como), Italy, and is currently marketed only in Italy. Defibrotide, obtained from CRINOS for investigational purposes and clinical trials in the United States, is available in ampules containing 200 milligrams for parenteral administration and in tablets containing 400 milligrams for oral administration.
Although verifiable data indicates that defibrotide, as a nucleic acid, is not toxic, mutagenic or harmful to fetal or embryonic development, maximum dosages of defibrotide administered to humans have been limited to either a body weight-dependent dose of 10-30 milligrams per kilogram, to an empirically established dose of 5.6 grams per day, intravenously, or a fixed dose of 800 milligrams per day, intravenously or by mouth. Coccoheri and Biagi (Cardiovascular Drug Reviews, 1991, 9(2): 172-196) report the highest dose of 2.4 to 5.6 grams daily which was given for three days only. These doses, which were based on previous animal studies, were administered empirically. Defibrotide was administered in conjunction with conventional therapy and produced modest advantage.
The pharmacodynamic effect obtained with oral administration of defibrotide is approximately one-half that of parenteral dosing (Fareed et al., 1988, Seminars in Thrombosis and Hemostatis--Supplement, 14: 27-37). The maximum dosages of oral defibrotide reported was 1600 milligrams per day. Even at these dosages, clinical improvement was much slower with the oral form of defibrotide than with the parenteral form.
Studies in human pharmacology have been conducted on the same non-dynamic, merely descriptive, principles as the pre-clinical studies, i.e., merely confirming the molecular events induced by defibrotide at pre-determined, set dose levels, uniformly assessed on the principles of using a "minimum efficacious dose." In healthy volunteers, 1200 mg/2 hours was reported to induce increases in the levels of 6-keto PgF.sub.1.alpha. and PgE.sub.2 (not confirmed subsequently by other investigators) (Gryzlewski R. J. et al., Eicosanoids, 1989, 2: 163-167), and 1200 mg/day for 2 weeks induced production of prostanoids and inhibition of arachidonic acid 5-lipoxygenase products, the latter known to contribute to pathogenesis and evolution of ischemic tissue damage.
The pro-fibrinolytic effect in healthy volunteers was confirmed by the administration of a single 400-600 mg dose with shortening only in euglobulin lysis time. Administration to peripheral obstructive vascular disease (POVD) patients in dose levels of 800 mg per day given orally, or 200, 400 and 800 mg per day given intramuscularly, displayed an additional effect of decreased PAI1 levels. Conversely, a more recent study failed to show significant activation of fibrinolysis in normal volunteers. A repeat study of POVD patients confirm significant defibrotide induced declines in PAI1 levels (Coccoheri and Biagi, 1991, Cardiovascular Drug Reviews, 9(2): 172-196). As a whole, the fibrinolytic effects in healthy volunteers were in general not reproducible, but in patients fibrinolytic effects were reproducible.
In direct opposition to its remarkable potentials in animal models and in vitro/ex vitro systems, published clinical studies with defibrotide have been notable for their modest-to-equivocal results in the respective areas of clinical application. One study continued intravenous defibrotide therapy for as long as three months ("Clinical Effectiveness of Defibrotide in Vaso-Occlusive Disorders and Its Mode of Actions", O. N. Ulutin, M.D. Thieme, Medical Publishers, Inc., Seminars in Thrombosis and Hemostasis--Supplement, Vol. 14, 1988). The majority of the remaining studies administered intravenous defibrotide for only two to three weeks, followed by the administration of oral defibrotide for a period of days to up to six months. Most studies ended at the discontinuation of defibrotide administration. Only one study followed patients for up to three years, although defibrotide was given for only three months ("Defibrotide: An Overview of Clinical, Pharmacology and Early Clinical Studies", Umberto Cornelli, M.D. and Marco Nazzari, M.D., Thieme Medical Publishers, Inc., Seminars in Thrombosis and Hemostasis--Supplement, Vol. 14, 1988). More recently, in a double-blind, randomized study conducted in peripheral arterial disease, defibrotide was administered orally for six months. Nearly all clinical studies (thus far) have focused on the agent's profibrinolytic and antithrombotic effects.
In summary, defibrotide has been shown in the art to have antithrombotic, thrombolytic, cytotropic, nephroprotective, platelet deaggregatory and anti-shock properties. These properties have been ascribed to its capacity to release PgI.sub.2 or its stable analogues from vascular endothelium. Defibrotide has also been shown to increase t-PA, decrease PAI1, increase protein S and C levels, increase ATIII (unconfirmed reports), increase platelet cAMP levels and, more recently, decrease endothelin-I levels and increase EDRF (endothelium derived relaxing factor) in in vitro models. The art has not recognized that alkalai salts of deoxyribonucleic acid, such as defibrotide, may be useful in the treatment of HIV infection.
In all studies heretofore reported, a particular patient's condition was assessed by using the subjective and objective clinical signs and symptoms of the patient. To date, laboratory results, such as various coagulation assays have been used only to determine the safety and efficacy of defibrotide treatment but not to tailor the therapeutic dose to the individual patient's disease entity or the disease severity or response to prior treatment. Escalating dose levels were never attempted and/or evaluated. Rather, the art has assumed that side-effects will occur when defibrotide is administered in amounts exceeding the "minimum efficacious dose."